The Tc17 Population Required For Sclerodermatous Chronic Gvhd Represents a Non-Cytolytic, Pluripotent Inflammatory Effector T Cell Lineage

IL-17 producing CD8⁺ T cells (Tc17) are a recently described effector T cell lineage. Donor T cell polarization is a critical factor influencing the severity and tissue distribution of graft-versus-host disease (GVHD) and the potency of graft-versus-leukemia (GVL) effects after bone marrow transplantation. However, CD8⁺ T cell lineage development during GVHD is not well understood. The recent shift to using G-CSF mobilized peripheral blood as a stem cell source for allogeneic stem cell transplantation (SCT) has increased the incidence of chronic GVHD (cGVHD) and we have recently reported that G-CSF promotes type-17 differentiation in both CD4 and CD8 T cells. Importantly, donor IL-17A, predominantly from CD8 T cells, mediates fibrotic skin pathology which manifests late after transplant as scleroderma (Hill GR, et al. Blood 2010, 116:819).

To study the donor Tc17 population, we utilized the IL-17A-^CrexR26R^eYFP fate-mapping reporter mouse that permanently labels any cell that has expressed IL-17A. In time course experiments, CD8⁺YFP⁺ Tc17 cells were rapidly induced in all lymphoid tissues and GVHD target organs examined (skin, liver and lung). This was a GVHD phenomena since no CD8⁺YFP⁺ T cells developed in syngeneic recipients. Importantly, the use of these transgenic cells confirmed that the majority of the IL-17 signal seen following re-stimulation with PMA/Ionomycin is not within the reporter population (i.e. is artefact). At day 7 post transplant Tc17 cells were the dominant IL-17 producing population and following re-stimulation, over 35% of the CD8⁺YFP⁺ cells co-expressed IL-17 and IFNγ seven days after BMT, while the remaining CD8⁺YFP⁺ cells had switched off IL-17 but maintained IFNγ production (i.e. had a Tc1 phenotype). By D21 only 5% of the reporter cells continued to produce IL-17, whereas IFNγ production was maintained, demonstrating significant plasticity within the CD8⁺YFP⁺ population. Thus Tc17 differentiation is an early and transient phenomenon with most cells going on to a Tc1 phenotype.

In order to understand the factors driving Tc17 differentiation during GVHD we utilized various cytokine neutralization strategies. The frequency of CD8⁺YFP⁺ Tc17 cells was significantly reduced in IL-6^–/– recipients (1.8% vs 5.1%, p=0.0002, WT vs IL-6^-/-) while IFNg neutralization (the dominant cytokine secreted early after BMT) resulted in a significant increase in frequency. CD8⁺YFP⁺ Tc17 development was dependent upon the presence of recipient DC as CD8⁺YFP⁺ frequencies were significantly reduced (5.2% vs 2.9%, p=0.0003) when host DC were depleted using diphtheria toxin (DT) treated CD11c-DTR recipients. CD8⁺YFP⁺ cells responded to stimulation with both mitogen and host-type DC by producing significantly increased levels of IL-17A (321 pg/mL ±11 pg/mL vs 3283 ± 147pg/mL, 24hrs p<0.001), IL-13 (252 ±12 pg/mL vs 542 ± 10pg/mL, 24hrs p<0.001), and TNF (1789 ±46 pg/mL vs 3140 ± 177pg/mL, 24hrs p<0.001) relative to CD8⁺YFP^neg cells, again suggestive of significant plasticity. These data demonstrate that recipient IL-6 production and recipient DC promote development of polyfunctional Tc17 cells.

Examination of lineage defining transcription factors in these populations revealed the CD8⁺YFP⁺ T cells expressed high levels of both RORγt and T-bet, the transcription factors associated with Th17 and Th1 differentiation respectively. Although IL-17 cytokine production ceased over time, the transcription factor signature was highly stable. In contrast the CD8⁺YFP^neg T cells expressed T-bet but not RORgT, suggesting these cells represent a different subset of cytotoxic Tc1 cells. This Tc1 phenotype was confirmed by their high level mRNA expression of the cytolytic proteins granzyme A and B which were minimally expressed in CD8⁺YFP⁺ Tc17 cells. The CD8⁺YFP⁺ Tc17 cells also failed to express granzyme B protein as determined by flow cytometry, in contrast to high expression by CD8⁺YFP^neg cells. Finally, the CD8⁺YFP⁺ cells were poorly cytotoxic in chromium release CTL assays relative to Tc1 effectors when assessed after BMT.

These data suggest that Tc17 differentiation is a very early and highly plastic differentiation program culminating in a non-cytolytic, pluripotent inflammatory donor T cell. Intervention to prevent this differentiation program, potentially via IL-6 or RORgt inhibition would be predicted to reduce cGVHD whilst maintaining therapeutic GVL effects.